Chapter 2

Question 1

Resolution

Answer 1

• is the smallest distance by which two objects can be separated and still be distinguished
• limits the ability of what we can see with a microscope

Question 2

Detection

Answer 2

is the ability to determine the presence of an object

Question 3

Magnification

Answer 3

is the increase in the apparent size of an image to resolve smaller separations between objects

Question 4

Microbial Shape

Answer 4

• Prokaryotic cell structures are generally simpler than those of eukaryotes
• Certain shapes of bacteria are common to many taxonomic groups
• Bacilli = rods
• Cocci = spheres
• Spiral forms = Spirochetes and Spirilla

Question 5

For electromagnetic radiation to resolve an object, certain conditions must exist

Answer 5

1. Contrast between object and its medium
2. Wavelength smaller than the object
3. Magnification

Question 6

Absorption

Answer 6

means that the photon’s energy is acquired by the absorbing object

Question 7

Reflection

Answer 7

means that the wavefront bounces off the surface of an object

Question 8

Refraction

Answer 8

is the bending of light as it enters a substance that slows its speed

Question 9

Scattering

Answer 9

occurs when the wavefront interacts with an object smaller than the wavelength of light

Question 10

empty magnification

Answer 10

magnification without increasing detail is called

Question 11

compound microscope

Answer 11

• a system of multiple lenses to increase magnification and produce an upright image
• Basic design contains 2 lenses = Ocular lens and Objective lens
• Total magnification = magnification of ocular multiplied by that of the objective

Question 12

wet mount

Answer 12

-simple way to observe microbes is to place them in a drop of water on a slide with a coverslip

Advantages:
-Observation of live cells in natural state

Disadvantages:

• Little contrast between cell and background
• Sample may dry out quickly

Question 13

Fixation

Answer 13

• cells are made to adhere to a slide in a fixed position with heat or chemicals
• usually kills the cells

Question 14

Staining

Answer 14

-cells are given a distinct color with a dye, which produces increased contrast

Question 15

simple stain

Answer 15

• adds dark color specifically to cells, but not to the external medium or surrounding tissue
• A commonly used stain is methylene blue

Question 16

differential stain

Answer 16

• stains one kind of cell but not another

- The most common differential stain is the Gram stain

Question 17

Gram stain

Answer 17

• differentiates between two types of bacteria
• Gram-positive bacteria retain the crystal violet stain because of their thicker cell wall
• Gram-negative bacteria do not

Question 18

Acid-fast stain

Answer 18

-carbolfuchsin used to stain Mycobacterium species

Question 19

Spore stain

Answer 19

malachite green used to detect spores of Bacillus and Clostridium

Question 20

Negative stain

Answer 20

• like india ink
• leaves a white area around the cells which i the carbohydrate layer
• carbohydrate layer use to protect the cells from our immune system
• colors the background, which makes capsules more visible

Question 21

fluorescence microscopy

Answer 21

• fluorescent molecules absorbs light of a defined wavelength, and then fluoresce by emitting light of lower energy, thus longer wavelength
• Can be used to stain specific cell types in complex samples or specific cellular components
• Can be used to follow expression of fluorescent reporter proteins such as GFP

Question 22

excitation wavelength

Answer 22

specimen absorbs light of a specific wavelength

Question 23

emission wavelength

Answer 23

emits light at a longer wavelength

Question 24

filters

Answer 24

• To limit incident light to the wavelength of excitation and emitted light to the wavelength of emission
• high-intensity light source shines through an excitation filter that transmits only the range of wavelengths to excite the fluorophore

Question 25

fluorophore

Answer 25

- is a fluorescent chemical compound Its cell specificity can be determined in at least 3 ways: 1) Chemical affinity for a cellular component 2) Labeled antibodies that bind to specific cellular components 3) DNA hybridization to specific DNA sequences

Question 26

Dark-Field Microscopy

Answer 26

- light stop is used to stop light from going directly through the sample to your eye - enables microbes to be visualized as bright objects against a dark background - Light shines on sample at an oblique angle - Only light scattered by the sample reaches objective - Makes visible objects below resolution limit - Flagella and Very thin bacteria

Question 27

Phase-Contrast Microscopy

Answer 27

- Superimposes refracted light and transmitted light shifted out of phase - Reveals differences in refractive index of different cell components as patterns of light and dark resulting from constructive and destructive interference - Can be used to view live unstained cells and internal cellular organelles of larger eukaryotic cells

Question 28

Electron Microscopy

Answer 28

Electrons behave like light waves - Very high frequency, which allows greater resolution - A few nanometers - Lenses are magnetic fields Sample must absorb or reflect electrons -Stained or coated with heavy metals (kills cells) Electrons are easily affected by matter - Electron beam and sample must be in a vacuum (cells must be dehydrated) - Samples must be very thin

Question 29

Transmission electron microscopy (TEM)

Answer 29

- Electrons pass through the specimen - Reveals internal structures - preparation- Embedded in a polymer for thin sections - Microtome with a diamond knife is used to cut very thin slices - Molecular samples can be sprayed onto a thin film supported by a copper grid - closely parallels the design of the bright-field microscope - light source is replaced by an electron source consisting of a high-voltage current applied to a tungsten filament, which gives off electrons when heated

Question 30

Scanning electron microscopy (SEM)

Answer 30

- Electrons scan the specimen surface - Reveals external features in 3D - preparation- specimen is not sliced but is coated with heavy metal - scattered and secondary electrons emitted from the sample are detected - electron beam is scanned across a specimen coated in gold, which acts as a source of secondary electrons

Question 31

X-ray crystallography

Answer 31

- For samples that can be crystallized, X-ray diffraction can determine the position of individual atoms in a molecule - Many organic molecules and proteins can be crystallized, but some do not produce suitable crystals - A beam of X-rays is shot at a crystallized sample - Many molecules are in identical conformation in the crystal - X-rays diffract according to position of atoms - Compute position of atoms from pattern of scattered X-ray - undergo mathematical analysis to generate molecular models